With respect to a gene of a virus whose host is a cat, a recombinant virus vector not naturally occurring has been created by artificially deleting, by genetic engineering technology, a part of the genome DNA of FHV-1, which is a herpesvirus belonging to an alphaherpes virinae subfamily of Herpesviridae and inducing viral nasal tracheitis in cats, and then introducing a foreign gene into the deleted region so as to express it in cells or animal bodies. It is known that such a recombinant vector upon infection of cells or animals produces not only a viral antigen derived from FHV-1 but also a product derived from the foreign gene, and upon inoculation into animals, gives immunity to the foreign gene product in addition to FHV-1 (N. Yokoyama et al., 1996, Arch. Virol. 141: 481-494; N. Yokoyama et al., 1996, Arch. Virol. 141: 2339-2351).
As an example of a such FHV-1 vector, a recombinant virus vector constructed by deleting a thymidine kinase (abbreviated hereinafter into TK) gene region and inserting a foreign gene into the deleted site is known (J. H. Nunberg et al., 1989, J. Virol. 63: 3240-3249; N. Yokoyama et al., 1995, J. Vet. Med. Sci. 57: 709-714). Such recombinant FHV-1 is confirmed to express not only FHV-1 protein but also a protein derived from the foreign gene introduced into the TK gene region, without damaging the ability to replicate the virus (G. H. Cole et al., J. Virol. 1990, 64: 4930-4938; R. C. Wardley et al., 1992, J. Gen. Virol. 73: 1811-1818). Further, Yokoyama et al. (N. Yokoyama et al., 1996, Arch. Virol. 141: 2339-2351) have reported that recombinant FHV-1 having a foreign gene inserted into the TK gene region is not pathogenic to cats, and a cat inoculated with the recombinant FHV-1 produces an antibody to a product of the foreign gene.
On one hand, an open reading frame 2 (ORF2) located downstream from the region of gC gene in the unique long (UL) region is known as an insertion site for a foreign gene other than the TK gene region in FHV-1 genome. Willemse et al. (M. J. Willemse et al., 1994, J. Gen. Virol. 75: 3107-3116) have reported that recombinant FHV-1 having a β-galactosidase-encoding gene (abbreviated hereinafter into LacZ) fragment as a foreign gene inserted into ORF2 maintains the same ability to replicate the virus as that of attenuated FHV-1. Further, it is also known that gene regions such as Us 8.5, gI and gE in the unique short (Us) region of the FHV-1 genome are not necessarily required for replication of FHV-1.
With respect to the recombinant FHV-1 having a foreign gene inserted into such a gene region, it is reported that recombinant FHV-1 having LacZ inserted into the US 8.5 gene region maintains the same ability to replicate the virus as that of attenuated FHV-1 (M. J. Willemse et al., 1995, Virology 208: 704-711), while it is reported that recombinant FHV-1 having LacZ inserted into gI and gE gene regions has significantly reduced the replication ability as compared with that of attenuated FHV-1 (M. J. Willemse et al., 1996, Vaccine 14: 1-5; M. D. Sussman et al., 1995, Virology 214: 12-20). In addition to recombinant FHV-1 using the Us gene region as an insertion site for a foreign gene, recombinant FHV-1 having a foreign gene inserted into ORF5 (nucleotide positions 5869-7113) of the FHV-1 genome has been proposed (Japanese Patent Application National Publication (Laid-Open) No. 2000-501927). However, the safety of these recombinant virus vectors to animals is not examined, and thus whether or not they are pathogenic to cats as the host is not evident. For reference, these insertion sites for foreign genes, together with a SalI map, are shown in a part of FIG. 1.
With respect to the recombinant FHV-1, several sites in the FHV-1 genome have been identified as insertion sites for foreign genes as described above, but there is no report on construction of a recombinant FHV-1 vector having a plurality of foreign genes inserted simultaneously into a plurality of gene insertion sites.
In consideration of application to a vaccine, a recombinant FHV-1 vector prepared by conventional techniques, including FHV-1 itself, can be used as a divalent vaccine, assuming that one type of foreign gene is inserted into essentially one gene insertion site. However, this does not meet a recently increasing demand for trivalent or more feline vaccines.
For maintaining the life cycle of virus, phosphorylation of viral protein is necessary, and cellular or viral protein kinases (protein phosphatase: serine/threonine kinase and tyrosine kinase) are considered to be involved in this phosphorylation. With respect to herpes simplex virus type 1 (HSV-1) and varicella-zoster virus (VZV) which like FHV-1, belong to the alpha-herpesvirus subfamily and Epstein-Barr virus (EBV) classified into the gamma-herpesvirus subfamily, the presence of a gene sequence encoding protein kinase in their virus genome is known, and an amino acid sequence encoded by the gene is concerved highly among the respective herpesviruses.
It is reported that in particular, catalytic domains I to VI in protein kinase are highly conserved among various herpesviruses (R. F. Smith, and T. F. Smith, 1989, J. Virol. 63: 450-455). A typical amino acid sequence of domain I in the catalytic domains of herpesvirus protein kinase is GXGXXGXV (SEQ ID NO.12)(X is a lowly conserved amino acid), and it has been found that such an amino acid sequence is highly conserved among HSV-1, VZV, EBV, human cytomegalovirus (HCMV) and human herpesvirus type 6 (HHV-6) (M. S. Chee, G. J. Lawrence, and B. G. Barrel. 1989, J. Gen. Virol. 70: 1151-1160).
In the prior art, however, there is no report on construction of a recombinant FHV-1 vector which except for TK gene-defective recombinant FHV-1 vector, is sufficiently attenuated to be safe to a cat, maintains a sufficient ability to replicate the virus produced as a vaccine virus and gives immunity simultaneously to three or more kinds of pathogenic gene products including FHV-1 itself. That is, in the known recombinant FHV-1 vector, the site into which a foreign gene can be inserted is limited essentially to one region in the FHV-1 genome, so that the cat inoculated with the recombinant FHV-1 is expected to have immunity to only one kind of foreign antigen besides FHV-1.
For providing the cat with immunity for protection against infection with, for example, 3 kinds of pathogenic microorganisms including FHV-1, it is therefore necessary to inoculate the cat with the recombinant FHV-1 having one kind of foreign gene inserted into it, and to further inoculate the cat with another kind of intended pathogenic microorganism or a vaccine consisting of an antigen derived therefrom. This, in production of vaccines, causes diversification of products and production processes, significantly increases production costs, and easily increases side effects due to inoculation with a plurality of vaccines consisting of pathogenic microorganisms.
On one hand, homologous genetic recombination between a vector virus and a naturally contagious virus in an animal is problematic upon the inoculation of a vector virus into an intended animal, and there is anxiety that the attenuated vector virus can acquire pathogenicity through the genetic recombination. To reduce such acquisition of pathogenicity by the attenuated vector virus through genetic recombination, it is necessary that gene mutations are induced in plural sites of the vector virus genome or foreign gene fragments are introduced into such sites, followed by homologous genetic recombination between the vector virus genome and the pathogenic virus genome, whereby the conversion of the vector virus into a pathogenic virus can be prevented unless gene sequences in the plural sites of the vector virus genome are simultaneously converted into pathogenic viruses. At present, however, the attenuated recombinant FHV-1 vector wherein a plurality of such gene regions are converted into gene sequences different from those of the pathogenic virus, or foreign genes are simultaneously introduced into a plurality of gene regions has never been created, mainly because the replication ability of the vector virus is lost.
For vaccines used in the prevention of feline viral infections or microbial infections, specifically vaccines for preventing infections with feline calicivirus, feline panleukopoenia virus, feline leukemia virus, rabies virus, FHV-1 and chlamydia, either the attenuated vaccines or inactivated vaccines are administered by a method of inoculation by injection. However, the method of inoculation by injection is not necessarily the best method because of the attendant troublesome procedures, and sharp pain for the animals. On the other hand, the method of inoculation via the mucosa, such as eye-dropping or nasal or oral inoculation, is superior to the method of inoculation by injection in many respects such as an easier inoculation procedure and induction of immunity via the mucosa without causing pain to the animals being inoculated.